KMS XINJIANG INSTITUTE OF ECOLOGY AND GEOGRAPHY，CAS
| 昆仑山北坡不同海拔豆科植物根瘤内生菌多样性及潜在根瘤菌特征分析 | |
| Alternative Title | Diversity of Endophytes within Root Nodules in Diverse Legumes and the Characteristic of Potential Rhizobium along an Elevation Gradient in the Kunlun Mountains |
| 庞金凤 | |
| Subtype | 硕士 |
| Thesis Advisor | 曾凡江 ; Brian P. Hedlund |
| 2019-06-30 | |
| Degree Grantor | 中国科学院大学 |
| Place of Conferral | 北京 |
| Degree Discipline | 工程硕士 |
| Keyword | 不同海拔高度 豆科植物 根瘤内生菌 潜在根瘤菌 寄主专一性 Elevation gradient Legumes Nodule endophytes Rhizobiales Host specificity |
| Abstract | 氮素是植物生长发育中至关重要的元素之一。 据统计， 豆科植物-根瘤菌共生体生物固氮为豆科植物贡献了超过 65％的氮元素，因此对于农牧业的发展及干旱区土壤和植被的管理具有重要意义。 然而， 目前对根瘤菌-寄主植物与生态环境之间的关系尚未被完全了解。 因此， 本研究以昆仑山北坡豆科植物根瘤为研究对象，通过分离纯化， 对从 16S rRNA 基因、 recA 基因序列物种多样性和关键生理特性实验中分离得到的内生菌进行了相关研究，揭示了昆仑山北坡区域豆科植物-根瘤菌和生态环境之间的内在联系和潜在根瘤菌的寄主专一性，为确定当地根瘤菌系统发育地位及合理利用昆仑山北坡根瘤菌资源库提供了理论依据。研究结果如下：（1） 在昆仑山北坡不同海拔（0-10 厘米， 10-20 厘米和 20-30 厘米） 土壤养分分析中，土壤的有机碳和全氮含量均随海拔升高显著增加。 全磷和土壤含水量显示出相似的趋势，但相关性不显著。相反，全钾和土壤 pH 均未显示出升高趋势。根际周围土壤显示出与之相似的海拔变化趋势，有机碳含量，全氮和全钾随海拔升高而增加，而 pH 和全钾未表现出明显的变化趋势。（2） 在豆科植物根瘤内生菌多样性特征的研究方面： 本研究中的豆科植物包括蝶形花亚科（6 种）和含羞草亚科（1 种）， 其中蝶形花亚科的根瘤内生菌多样性更高。在分离出的内生菌中， 芽孢杆菌在所有海拔高度的全部宿主植物中占主导地位，其次是根瘤菌和假单胞菌。 从五个不同的海拔的五个不同的宿主豆科植物中获得 39 个潜在根瘤菌。 Spearman 相关分析表明， Pseudomonas 相对丰度与根际周围土壤全钾含量呈显著负相关关系，而 Bacillus 相对丰度与昆仑山样方土壤全磷含量、全钾含量以及 pH 呈显著负相关关系。（3） 在潜在根瘤菌系统学研究方面：通过 recA 基因测序鉴定出的 36 个根瘤菌菌株，分别属于 9 个不同的系统发育分支， 但均属于 Rhizobiales。源自 recA 基因的系统树与 16S rRNA 基因的系统树基本一致。根据 recA 系统发生学，根瘤菌分离株属于 Agrobacterium， Allorhizobium， Bosea， Ensifer， Microvirga，Pararhizobium， Phyllobacterium， and Rhizobium，分离株在物种水平上具有很高的多样性，其中 Ensifer 和 Rhizobium 是最大的群体。（4）对 39 株潜在根瘤菌的关键表型特征测定中发现， 所有菌株都可以在 Ashby’sMannitol Agar（AMA）无氮培养基上正常生长，表明它们可以固定大气中的氮；所有菌株均在 25℃下生长良好，但无法在 60℃下生长； 从高海拔（> 2600 m）分离出的许多菌株（10/13）能够在 4℃下生长，而从低海拔分离的只有一半（13/26）可以在 4 ℃下生长；同时， 这些菌株具有广泛的异养活性，大量菌株能够利用麦芽糖（37/39），蔗糖（35/39），肌醇（35/39）和淀粉（31/39）。相比之下， 极少数（7/39） 分离株可以利用果糖，没有菌株利用丙酸酯， 大多数（30/39） 菌株能够从甘露醇培养基中生产酸。大多数菌株能够以单一的 L-氨基酸或硝酸盐作为唯一的氮源生长， 但是，只有极少数（2/39）能够利用铵。所有测试的菌株都能够在 3％NaCl 条件下正常生长，而只有少数菌株在 7-8％NaCl 下正常生长； 几乎所有菌株的 pH 生长范围在 6-10。 结果表明， 不同潜在根瘤菌在相同的生长条件下生长状况存在差异， 同一菌株的生长状况也因原有生长环境的不同即使在相同的生长条件下也表现不同。 |
| Other Abstract | Nitrogen is one of the most important elements in plant growth and metabolism.According to statistics, the bio-nitrogen fixation of leguminous-rhizobial symbiontscontributes more than 65%, so it has great significance for agriculture and animalhusbandry and arid soil and vegetation. However, the relationship between rhizobium,host plants and the ecological environment is not fully understood, understanding andsystematically mastering the host specificity and intrinsic relationship betweenregional legumes, rhizobium and ecological environment will provide a scientificreference for determining the developmental status of local rhizobial phylogeny todiscover new excellent nodules adapted to the local ecological environment. Thegermplasm resources and rational use of the rhizobial resource pool provide atheoretical basis. In this study, we investigated the root nodules of leguminous plantson the northern slope of Kunlun Mountains, the isolated endophytes were studied by16S rRNA gene, recA gene and key physiological characteristics experiments. Theresults show that:1) In bulk soils, total organic carbon (TOC) and total nitrogen (TN) both increasedsignificantly with altitude at each of three soil depths, 0-10 cm, 10-20 cm, and 20-30cm. Phosphorous (P) and soil moisture content showed similar trends, but therelationships were not statistically significant. In contrast, neither potassium (K) norsoil pH showed elevational trends. Rhizosphere soils showed similar elevationaltrends, with TOC, TN, and K generally increasing with elevation. On the other hand,pH and K did not show significant trends.2) The host legumes in this study included members of the Papilionoideae (sixspecies) and the Caesalpinioideae (1 species). Accordingly, a higher diversity ofendophytic bacteria was isolated from root nodules of Papilionoideae. Among theisolated endophytes, Bacillales (genus Bacillus) dominated in all host legumes, at all elevations, followed by Rhizobiales and Pseudomonadales. 39 isolates belonged tothe Rhizobiales, and these were considered to be potential rhizobia. Rhizobialesisolates were obtained from five different host legumes and five different elevations.Spearman’s rank correlation analysis showed that the relative abundance ofPseudomonas among the isolates was negatively correlated with total potassiumcontent of rhizosphere soil, the relative abundance of Bacillus among the isolates wasnegatively correlated with total potassium content， total phosphorus content and pH ofbulk soil.3) 36 Rhizobiales strains that could be identified by recA gene sequencing formednine phylogenetic clusters. These belonged to Rhizobiales. In general, thephylogenetic trees derived from the recA gene were consistent with those derivedfrom the 16S rRNA gene. According to the recA phylogeny, the Rhizobiales isolatesbelonged to the genera Agrobacterium, Allorhizobium, Bosea, Ensifer, Microvirga,Pararhizobium, Phyllobacterium, and Rhizobium. The recA phylogeny suggested alarge diversity at the species level, with Ensifer and Rhizobium as the largest groups.4) The 39 Rhizobiales isolates were tested for key phenotypic traits that might beimportant to their ecology. All strains grew normally on Ashby’s Mannitol Agar(AMA) nitrogen-free medium, suggesting they can fix atmospheric nitrogen. Allstrains grew well at 25 °C, but were not capable of growth at 60 °C. Many (10/13) ofthe strains isolated from high elevation (>2,600 m) were capable of growth at 4 °C,whereas only half (13/26) from low-altitude sites grew at 4 °C. In general, the strainshad broad heterotrophic activity, with large number of strains using maltose (37/39),sucrose (35/39), inositol (35/39), and starch (31/39). In contrast, very few isolatesused fructose (7/39) and no strains used propionate. Most strains (30/39) were capableof producing acid from mannitol. Most strains were able to grow with single L-aminoacids or ammonium as the sole nitrogen source; however, very few (2/39) were able toutilize nitrate. All tested strains were able to grow normally on 3% NaCl, while only a ew strains grew normally at 7-8% NaCl. Phyllobacterium isolates from highelevation had the highest tolerance for high salinity. Nearly all strains had a pHgrowth range of 6 to 10. It shows that the growth status of different potentialrhizobium differs on the same growth conditions, and the growth status of the samerhizibium varies to different growth conditions due to the original growthenvironment. |
| Subject Area | 生物工程 |
| Language | 中文 |
| Document Type | 学位论文 |
| Identifier | http://ir.xjlas.org/handle/365004/15351 |
| Collection | 中国科学院新疆生态与地理研究所 研究系统 |
| Affiliation | 中国科学院新疆生态与地理研究所 |
| First Author Affilication | 中国科学院新疆生态与地理研究所 |
| Recommended Citation GB/T 7714 | 庞金凤. 昆仑山北坡不同海拔豆科植物根瘤内生菌多样性及潜在根瘤菌特征分析[D]. 北京. 中国科学院大学,2019. |
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